Departamento de Biotecnología, Universidad Autónoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco 186, Col. Vicentina, México D.F., 09340, Ciudad de México, Mexico.
Departamento de Ciencias de la Alimentación, Universidad Autónoma Metropolitana-Lerma, Av. Hidalgo Poniente 46, Col. La Estación, 52006, Lerma de Villada, Estado de México, Mexico.
Extremophiles. 2021 May;25(3):311-317. doi: 10.1007/s00792-021-01230-3. Epub 2021 May 3.
Fucosylated oligosaccharides present in human milk perform various biological functions that benefit infants' health. These compounds can be also obtained by enzymatic synthesis. In this work, the effect of the immobilization of α-L-fucosidase from Thermotoga maritima on the synthesis of fucosylated oligosaccharides was studied, using lactose and 4-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as acceptor and donor substrates, respectively, and Eupergit CM as an immobilization support. The enzyme was immobilized with 90% efficiency at pH 8 and ionic strength of 1.5 M. Immobilization decreased enzyme affinity for the donor substrate as shown by a 1.5-times higher K value and a 22-times decrease of the k/K ratio in comparison to the unbound enzyme. In contrast, no effect was observed on the synthesis/hydrolysis ratio (r/r) when α-L-fucosidase was immobilized. Also, the effect of initial concentration of substrates was studied. An increase of the acceptor concentration improved the yields of fucosylated oligosaccharides regardless enzyme immobilization. The synthesis yields of 38.9 and 40.6% were obtained using Eupergit CM-bound or unbound enzyme, respectively, and 3.5 mM pNP-Fuc and 146 mM lactose. In conclusion, α-L-fucosidase from Thermotoga maritima was efficiently immobilized on Eupergit CM support without affecting the synthesis of fucosylated oligosaccharides.
人乳中存在的岩藻糖基化寡糖具有多种有益于婴儿健康的生物学功能。这些化合物也可以通过酶合成获得。在这项工作中，研究了嗜热栖热菌α-L-岩藻糖苷酶固定化对岩藻糖基化寡糖合成的影响，分别以乳糖和 4-硝基苯基-α-L-岩藻吡喃糖苷（pNP-Fuc）作为受体和供体底物，以 Eupergit CM 作为固定化载体。在 pH 8 和离子强度为 1.5 M 的条件下，酶的固定化效率达到 90%。固定化降低了酶对供体底物的亲和力，表现为 K 值增加了 1.5 倍，k/K 比值降低了 22 倍，与未结合的酶相比。相比之下，当α-L-岩藻糖苷酶固定化时，对合成/水解比（r/r）没有影响。此外，还研究了初始底物浓度的影响。增加受体浓度无论酶是否固定化都能提高岩藻糖基化寡糖的产率。使用 Eupergit CM 结合或未结合的酶，在 3.5 mM pNP-Fuc 和 146 mM 乳糖的条件下，分别获得了 38.9%和 40.6%的合成产率。总之，嗜热栖热菌α-L-岩藻糖苷酶在不影响岩藻糖基化寡糖合成的情况下，有效地固定在 Eupergit CM 载体上。
